Na+/H+ exchanger is a broadly distributed transporter that plays a central role in a variety of cellular processes including: (l) cell volume regulation, (2) intracellular pH regulation, (3) normal and abnormal cell growth and differentiation, and (4) fluid-electrolyte and acid-base balance at the level of the organism. Although less known, the K+/H+ exchanger also seems to be involved in similar processes. The complexity of the controlling mechanism and the possibility of multiple combinations of sensors and isoforms of the exchanger, have been used to explain in part the versatility of the exchangers and their involvement in many physiological and pathophysiological processes. In order to understand the fundamental role of these transporters, we need a better understanding of their basic characteristics and regulation at the cellular leveL In this regard, the Amphiuma red blood cells has become one of the ideal model system in which the physiology, pharmacology and biochemistry of the exchangers can be studied. It has been previously demonstrated that in Amphiuma red blood cells the Na+/H+ and K+/H+ exchangers can be selectively activated by changes in cell volume or intracellular pH. In addition, it has been proposed in the past that the same transport moiety is responsible for the two types of exchangers. A model, involving the Ca++-calmodulin and protein kinase-C pathways, have been proposed to explain this coordinated and volume sensitive activation. In this model, net phosphorylation of the carrier, by the combined activity of protein kinase C and a calyculin sensitive phosphatase, results in the activation of a non-selective mode of the exchanger. In this mode the carrier is capable of transporting Na+, K+, and H+ in either direction resulting in a net Na+/K+ exchange. A second event associated with the volume perturbation, and involving the modulation of Ca++-calmodulin, is responsible for conferring ion selectivity. Swelling of the cells will increase Ca++-calmodulin levels which results in activation of the K+/H+ exchange mode while shrinkage of the cells results in the activation of the Na+/H+ exchange mode. The main goal of the experiments proposed here is to test part of this hypothesis by establishing a direct correlation between the levels of activity of the regulatory signals and the activation of the transport response. We will analyze the changes in the intracellular levels of protein kinase-C and Ca++-calmodulin in response to the pH and volume signals. The study will be focussed on the role of these messengers in the coordination, activation, and deactivation of both Na+/H+ and K+/H+ exchangers. In addition, the activity of these messengers will be also manipulated with the use of pharmacological tools like phosphatase and kinase inhibitors, phorbol esters and calcium ionophore. Finally, the possible involvement of other protein kinases in the activation of the exchangers by changes in volume and pH, will also be studied.